As described in the goals and objectives section of this report, this project consists of four specific aims: Developing Tools for Experimental Analysis of Vascular Development in the Zebrafish The development of new tools to facilitate vascular studies in the zebrafish has been an important ongoing aim of this project. In previous work we (i) developed a widely used confocal microangiography method (ii) compiled an atlas of the anatomy of the developing zebrafish vasculature, (iii) generated a variety of transgenic zebrafish lines expressing different fluorescent proteins within vascular or lymphatic endothelial cells, making it possible for us to visualize vessel formation in intact, living embryos, and (iv) developed methodologies for long-term multiphoton confocal timelapse imaging of vascular development in transgenic fish. We are currently developing many new transgenic lines useful for in vivo vascular imaging as well as for in vivo blood or lymphatic endothelial-specific functional manipulation of signaling pathways involved in vascular specification, patterning, and morphogenesis. Notably, we have generated RiboTag and AgoTag transgenic zebrafish for high-throughput whole genome tissue-specific profiling of gene and microRNA expression, and we have demonstrated that these transgenic lines can be used to perform in vivo profiling of vascular signaling pathways. We are also developing transgenic tools for tissue-specific proteomic profiling in the vasculature. Genetic Analysis of Vascular Development Previously, we have used forward-genetic ENU mutagenesis screens in transgenic zebrafish to generate, identify, and characterize many new zebrafish mutants affecting the formation of the developing vasculature. We identified and positionally cloned mutants with phenotypes including loss of most vessels or subsets of vessels, increased sprouting/branching, and vessel mispatterning. These mutants have resulted in numerous important discoveries related to endothelial specification, arterial differentiation, vascular patterning, and VEGF signaling, to mention only a few. Recent publications include studies on mutants in RECK and aminoacyl-tRNA synthetases affecting VEGF signaling and angiogenesis, on the role of Rac signaling in angiogenesis, and on targeting of phosphoinositide recycling as an anti-angiogenic cancer approach. Many of our current efforts are directed at understanding the cellular and molecular basis for the defects in mutants we have obtained affecting blood or lymphatic vessel formation or vascular integrity. Analysis of Vascular Specification, Patterning, and Morphogenesis We have previously used multiphoton time-lapse imaging to characterize patterns of vessel assembly throughout the developing zebrafish, and used molecular and experimental analysis understand how this pattern arises and what cues guide vascular specification, differentiation, and network assembly during development. Our discoveries have included evidence that neuronal guidance factors play an important previously unknown role in vascular guidance and vascular patterning. Our current work includes projects aimed at (a) studying the specification, differentiation, and patterning of vascular smooth muscle in the zebrafish, making use of newly developed transgenic tools, (b) understanding the role of intracellular signaling substrates in regulating vascular endothelial signaling, (c) exploring the role of RhoA signaling in vessel formation and vessel integrity, (d) characterizing the complex assembly and patterning of the vasculature of the pectoral fin, an analogous structure to mammalian forelimbs (arms). Emergence of hematopoietic cells Kctd15 may regulate blood formation in zebrafish. In a collaboration involving Alison Heffer, Aniket Gore, Valeria Zarelli, Matthew Brooks, Linn Gieser, Igor Dawid and Brant Weinstein we followed up on our earlier analysis of the biological functions of kctd15 in zebrafish (Heffer et al., PLoS One 12(12):e01891622017) by carrying out RNA-Seq analysis at several stages of kctd15 mutant and wildtype zebrafish. The results are deposited under the GEO accession number GSE136448. Related data are available under GEO accession number GSE136449. A surprising observation was that genes with GO terms related to blood development such as blood, intermediate cell mass of mesoderm, and blood island are significantly overexpressed in mutant as compared to wildtype embryos at 2 days post fertilization. Preliminary analysis indicated that blood cells in mutant embryos are morphologically abnormal. The apparent role of Kctd15 proteins in the regulation of blood formation, not previously noticed, may be a worthy subject for future studies.